by Group A
Roll no. 1 - 14
LABORATORY DIAGNOSIS
OF PULMONARY
TUBERCULOSIS
SPECIMEN COLLECTION
• In pulmonary TB: Sputum
-according to NTEP 2 sputum samples are collected,
-according to WHO and CDC, 3 sputum specimen are
collected
• When sputum is not available, laryngeal aspirates,
or bronchial washings
In children - gastric aspirate
Digestion,
decontamination and
concentration of specimen:
• Modified Petroff’s method (4% NaOH)
• NALC (N-acetyl-L-cysteine) (2% NaOH)
Microscopy
• Ziehl-Neelsen (ZN) technique
Interpretation:
No. of AFB seen OIF to be screened Grading Result
No AFB in 100 OIF 100 0 -ve
1-9/100 OIF 100 Scanty +ve
10-99/100 OIF 100 1+ +ve
1-10/OIF 50 2+ +ve
>10/OIF 20 3+ +ve
Positive-long slender, beaded, less uniformly stained red color acid-fast bacilli.
Negative- At least 100 oil immersion fields
should be examined for 10–15 minutes before giving a negative report
Other Methods
• Kinyoun’s cold acid-fast staining
• Fluorescent staining- Auramine rhodamine
stain
Culture
Solid media
• Lowenstein Jensen (LJ) medium— shows rough, tough
and buff colored colonies in 6–8 weeks
• Other- Dorset medium
Tarshis medium
Loeffler’s medium
Liquid media –used for drug susceptibility more than
culture
• Dubos’ medium
• Middle brooke’s medium
Culture Identification
• Automated identification - by MALDI-TOF
• MPT 64 antigen detection - by ICT
Automated System
• BACTEC MGIT
• BacT/ALERT
- Slowly replacing solid culture methods becaue
of rapid indication of growth.
Biochemical tests
• Niacin test
• Aryl sulphatase test
• Catalase peroxidase test
• Nitrate reduction test
• Amidase test
• Pyrazinamidase test
Animal Inoculation
• Guinea pigs- IM injection into the thigh of 2
healthy guinea pigs about 12 weeks old
Progressive loss of weight is the indication of
infection
Molecular methods
• PCR detecting IS6110 gene
• LCR (Ligase Chain Reaction)
• GeneXpert
Serology
• By antigen-antibody detection
• Not recommended by WHO due to low
sensitivity and cross reactivity with other
mycobacteria
Immunodiagnosis
• Tuberculin test
Mantoux test
0.1 ml of PPD intradermal injection on forearm.
Measure induration after 48-72hrs.
Diameter of induration Interpretation
10mm or more +ve
5mm or less -ve
6-9mm equivocal
Reference
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